The expression of a heterologous nucleotide sequence in a plant cell is impacted by regulatory nucleic acids. Promoters and terminators are two types of regulatory elements that impact expression of such operably linked sequences. Promoters are vital molecular tools that have been applied widely in plant biotechnology to control the expression of introduced genes. A promoter is a nucleic acid sequence to which RNA polymerase must bind if it is to transcribe the linked gene into messenger RNA and ultimately produce protein. A promoter may affect a structural gene operationally associated with the promoter in different ways. For example, it may enhance or repress expression of an associated structural gene, subject that gene to developmental regulation, or contribute to the tissue-specific regulation of that gene. There are different types of promoters used dependent upon the function desired. Constitutive promoters provide for expression throughout all tissues of the plant, where tissue preferred promoters will express at a higher rate in a (or a few) select tissue of the plant. Inducible promoters are those which induce the regulatory affect of the promoter in response to a stimulus, which can be, for example, chemical, temperature, stress, wounding or other stimuli. The linked nucleotide sequence can perform any of a wide variety of functions desired, whether it is repressing or initiating expression of a trait or protein of interest, providing for over-expression, modifying metabolic and developmental pathways within the plant tissue, or the like.
Several promoters of plant and plant pathogen (bacterial and viral) origin have been used to direct transgene expression in plants. Prominent examples include the French bean beta-phaseolin promoter (Bustos et al., 1989), the mannopine synthase promoter of Agrobacterium tumefaciens (Leung et al., 1991), and the 35S promoter of cauliflower mosaic virus (Guilley et al., 1982). These and several other promoters in widespread use in plants were originally developed and utilized in dicot species. Despite the desire to identify constitutive promoters capable of driving a relatively high level of gene expression in most tissues of the plant, there remain few to choose from and there is an ongoing need to identify promoters for use in expressing linked sequences.
Terminator sequences also play an important role in regulation of gene expression. The 3′ terminus of an isolated nucleotide sequence is the site as which transcription stops. A terminator region can be native with the promoter used, can be native with the linked heterologous sequences or derived from another source.
Ubiquitin is a 76 amino acid polypeptide found in all eukaryotes and has been studied for its role in a wide range of cellular functions. Promoters of the ubiquitin gene have been isolated. For example, in U.S. Pat. Nos. 5,510,474, 5,614,399, 6,054,574 and 6,020,190 to Quail is described ubiquitin promoters which include a heat shock element and intron. Jilka et al. describe another maize ubiquitin type promoter at U.S. Pat. No. 6,977,325. Xia et al. identified a soybean genomic clone containing a ubiquitin gene (Xia et al., 1994). Analysis of the nucleotide sequence of this clone revealed the presence of a translational start site, a translational stop codon, a 915-bp open reading frame encoding polyubiquitin (arranged as three tandem 228-bp head-to-tail repeats and a fourth terminal repeat containing 231 bp), a putative polyadenylation signal motif. However, no transcription initiation site was identified. These sequences are reported at GenBank accession numbers D16248.1 and D2823.1. Also, Finer et al. have discussed analysis of a soybean ubiquitin promoter, but did not provide a sequence (Finer et al., 2006). Thus there remains a need for the identification of structure and function of promoters and terminators, and a high expressing regulatory region would be especially desirable.
All references cited herein are incorporated herein by reference.